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View additional product information for CytoTune™-iPS 2.0 Sendai Reprogramming Kit - FAQs (A16518, A16517)
54 product FAQs found
The shelf life for this kit is 3 years from the date of manufacture. Both the date of manufacture and the date of expiration are stated on the lot-specific Certificate of Analysis (COA).
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The shelf life for the CytoTune-iPS 2.0 Sendai Reprogramming Kit is 2 years when stored at -80 degrees C with no repeated freeze-thaw cycles.
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We recommend reprogramming patient cells at the earliest passage possible. However, it is important to have the cells growing and healthy, which can take between 1-4 weeks. The cells are usually ready to reprogram once they have gone through a total of 3-4 passages.
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Yes. Initial experiments with fibroblasts have shown that scaling down to a 12-well or 24-well works, but at a potentially reduced efficiency. Cell seeding densities may need to be optimized.
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We recommend seeding approximately 50,000-100,000 fibroblast cells per well of a 6-well to yield 200,000-300,000 cells two days later. This equals a confluency of 50-80% on the day of transduction. Overly confluent cells will result in a decreased transduction efficiency. When cells are overly confluent (>80% confluent), we recommend re-plating the cells to achieve a range of 50-80% confluency on the day of transduction.
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The CTS CytoTune-iPS 2.1 Sendai vector formulation does not contain the bovine serum albumin carrier protein. Additionally, the preparation contains no animal-derived components at the primary component level. Each virus (i.e., KOS, L-Myc, and Kfl4) comes in a volume of 200 µL instead of 100 µL. The titer of the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit can range from 0.5 x 10E8 to 2.0 x 10E8 virus particles per mL whereas the titer of the CytoTune-iPS 2.0 Sendai Reprogramming Kit is typically around 1.0 x 10E8 virus particles per mL.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of CD34+ blood cells. For CD34+ cells, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 39-44). On Day 3, you can utilize rhVTN-N (Cat. No. A14700), Geltrex (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, reprogrammed CD34+ cells should be fed every-other-day with StemFlex Medium.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.
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Valproic acid is typically thought to enhance reprogramming with integrating viral systems such as lentivirus. It is unlikely to enhance CytoTune reagent reprogramming since Sendai virus is a non-integrating RNA virus.
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The Invitrogen CytoTune-iPS Sendai 2.0 Reprogramming Kit contains a temperature sensitive mutant of c-Myc and KOS that facilitates the clearance of these vectors. To clear c-Myc and KOS, incubate the iPSCs at 38-39 degrees C for 5 days. One caveat is that given the sensitive nature of iPSCs, only perform this temperature shift if Sendai virus is in your iPSC lines after more than 10 passages, and you have performed RT-PCR to show that the Klf4 vector is absent from your cells (this vector does not have temperature-sensitive mutations). Then you can perform temperature shift to remove the c-Myc and KOS vectors.
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Depending on your cell type, you should expect to see some cytotoxicity 24-48 hours post-transduction, which can affect >50% of your cells. This is an indication of high uptake of the virus and is caused by the expression of exogenous genes. We recommend that you continue culturing your cells and proceed with the protocol.
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AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs) and Embryonic Carcinoma Cells (ECCs). While AP is expressed in most cell types, its expression is highly elevated in PSCs. Therefore, AP staining has been used to differentially stain PSCs to easily distinguish them from mouse embryonic fibroblasts (MEFs) used as feeders and parental fibroblasts commonly used in reprogramming experiments.
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The TaqMan Sendai Gene Expression Assays are compatible with both CytoTune 2.0 and CytoTune 2.1, with one exception. The CytoTune 2.0 kit contains a vector which expresses c-Myc, while the CytoTune 2.1 kit contains a vector which expresses L-Myc. As such, the relevant assays should be used in conjunction with each kit (e.g. SEVCYMC for CytoTune 2.0, and SEVLMYC for CytoTune 2.1). All other assays are compatible with both kits.
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We do not have such primers for use in endpoint RT-PCR. However, we do offer validated TaqMan Gene Expression assays for each of the exogenous reprogramming genes (https://www.thermofisher.com/order/genome-database/browse/gene-expression/keyword/sendai+taqman). These assays are designed to specifically detect only expression of the exogenous genes found on the Sendai vectors, and will not cross-react with expression of the endogenous versions of these genes. Additionally, these assays can be used to detect the presence of each of the CytoTune Sendai vectors, for determination of clearance of the vectors from established iPSCs.
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For the Invitrogen CytoTune 2.0 kit, which contains 3 vectors (KOS, cMyc, Klf4), both the cMyc and the KOS vector can be cleared by a temperature shift to 39 degrees C. The temperature shift will be most effective for the cMyc vector, but should still work for the KOS vector.
Note: Before performing a temperature shift, first verify that the individual Klf4 vector has cleared. The Klf4 vector will not be cleared by a temperature shift, and performing the shift while the Klf4 is still present could cause problems.
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You can confirm the presence of the Sendai virus in your cells by several methods, including staining with anti-Sendai virus antibody, performing endpoint RT-PCR using the primer sets listed on page 50 in the CytoTune-iPS 2.0 Sendai Reprogramming Kit User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf), or real-time RT-PCR using the TaqMan Sendai Gene Expression assays, also listed on page 50 in the CytoTune-iPS 2.0 Sendai Reprogramming Kit User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
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If you want to use the EmGFP Reporter with reprogramming, it must be added at the time of reprogramming. Cells infected with Sendai virus will most likely be refractive to further infection. Therefore, do not try to add Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit to cells already transduced with Invitrogen CytoTune-EmGFP Sendai Fluorescence Reporter or vice versa.
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It can take as few as five or as many as fifteen passages for the virus to clear from the cell. In rare cases, Sendai sequences can persist indefinitely. Clearance rate is clone-dependent and can be confirmed by PCR or by anti-Sendai antibody. For more information about generating vector-free iPSCs, please refer to the user manual (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
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iPSCs must be monitored and growth medium must be replaced daily in order to maintain a healthy culture. In general, iPSC colonies should be passaged when the cells reach 70-80% confluence or when most of the colonies are larger than 700 µm. Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
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We have used this combination of primary and secondary with no problems. IgG secondary antibodies will cross-react with IgM primary antibodies, since IgM share the same kappa light chains as IgG.
The IgG secondary antibodies that are listed in manuals are available, and they will work against IgG primary antibodies, as well as IgM primary antibodies, and others. For that reason, we typically have those IgG secondary antibodies on hand, and use them in most applications. That is the main reason it was recommended.
Please note that the Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued.
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The iPSC colonies can be easily visualized using alkaline phosphatase stain, such as the Alkaline Phosphatase Live Stain (Cat. No. A14353). In addition, reprogrammed colonies can be selected utilizing live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types. Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/AP_Live_Stain_man.pdf).
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The expected morphology of iPSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. If you do not observe this morphology or the number of colonies observed is low, then the MOI used for transduction may need to be increased.
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We do not recommend using VPA with the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit.
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The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit offers reprogramming efficiencies in the range of 0.02-1.2% with BJ fibroblasts. The reprogramming efficiency may vary for other cell types.
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You can cryopreserve iPSCs just as you would cryopreserve any pluripotent stem cells. Growth medium with 10% DMSO is recommended for freezing, or our PSC cryopreservation kit (Cat. No. A2644601) can be used
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iPSC colonies will begin to form roughly 3 weeks post-transduction. Only one application of the vectors is required for successful reprogramming, enabling selection of iPSC colonies 21-28 days after transduction.
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Avoid repeated freezing and thawing of the reprogramming vectors. Viral titers can decrease significantly with each freeze-thaw cycle and are not guaranteed for kits that have been refrozen or thawed.
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The reprogrammed cells can be grown in standard iPSC culture medium. We recommend either Gibco KnockOut Serum Replacement (KSR)-supplemented medium in a feeder-dependent culture, or feeder-free in Gibco Essential 8 Medium. Please refer to the user manual (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf) for the full protocol.
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If you want to reprogram PBMCs with Invitrogen CytoTune 2.0 under feeder-free conditions, you should follow the existing Invitrogen CytoTune 2.0 PBMC protocol, but plate onto Gibco Vitronectin or Geltrex matrix on day 3 instead of MEF, and then transition over to Gibco Essential 8 on Days 7-8 instead of Gibco KnockOut Serum Replacement-based PSC medium.
Efficiencies are typically lower than with feeder-dependent conditions, but you should still get some colonies.
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We recommend first to try increasing the MOI of hKlf4 only. For example: go from 5:5:3 to 5:5:6. If optimization is still required, then increase the MOI of KOS and hc-Myc. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: go from 5:5:3 to 10:10:3 or 10:10:6.
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An MOI of 5:5:3 (KOS, hc-Myc, hKlf4) is recommended for most cell types. The virus titer varies from lot to lot; the required volume for each MOI is listed on the Certificate of Analysis (CoA) for each lot of product. You may also choose to optimize your MOI as this may vary depending on the cell type. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: if KOS is 4, then hc-Myc must also be 4.
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MOI (multiplicity of infection) describes the ratio of viral particles to cells. The three vectors in the Invitrogen CytoTune 2.0 Kit should each be added to the cells based on an MOI recommendation. We recommend the following MOIs as a starting point, and adjustments can be performed if reprogramming efficiency is not optimal:
- Invitrogen CytoTune 2.0 KOS: Starting MOI of 5
- Invitrogen CytoTune 2.0 hc-Myc: Starting MOI of 5
- Invitrogen CytoTune 2.0 hKlf4: Starting MOI of 3
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One kit (one tube of each vector, i.e., 3 tubes) is sufficient for a minimum of 5 wells of a 6-well dish at MOI of 5:5:3 (KOS, hc-Myc, hKlf4) with a recommended plating density of 2 x 10E5 to 3 x 10E5 cells/well for human dermal fibroblasts. The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.
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We recommend using the virus only once as viral titers decrease significantly with each freeze thaw cycle.
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Yes, you must use all three reprogramming vectors together. Omitting one or two of the vectors will likely result in little or no reprogramming.
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Upon receipt, these kits should be stored at -80 degrees C.
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The virus does get passed on to daughter cells, and gradually over time its concentration diminishes. The virus can't leave the cells and infect new cells though, because the fusion gene has been deleted from the viral genome.
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Although humans are not a natural host for SeV, and the virus is non-pathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash.
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Yes, we have successfully induced iPSCs from mouse embryonic fibroblasts using our CytoTune-iPS 2.0 Sendai Reprogramming kit.
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Yes. Please go here for a protocol (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols.html) or publication (http://www.thermofisher.com/us/en/home/life-science/stem-cell-research/stem-cell-research-learning-center/stem-cell-research-resource-library.html).
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Our reprogramming kits have been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, and peripheral blood mononuclear cells (PBMCs). For a current list of publications citing the cell types validated using this method, go here (http://www.thermofisher.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming/cytotune-publications.html).
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Integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes. Traditional technologies used for reprogramming (e.g., lentivirus, retrovirus) integrate into the genome of the target cells. The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications. Furthermore, the integration could occur in a critical region of the genome, causing problems with unrelated developmental processes.
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The Sendai virus vectors in the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit are based on a modified, non-transmissible form of SeV, which has the fusion protein gene (F) deleted. The viral vectors maintain full infectivity to a wide range of cells; however they are no longer capable of producing infectious particles from infected cells because the viral genome lacks the F gene. The Sendai virus vectors contain transgenes that will express factors hOct3/4, hSox2, hKlf4, and hc-Myc. After transduction, the viral vectors will cause the cells to express these four genes, resulting in reprogramming
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Sendai virus, also known as Hemagglutinating Virus of Japan (HVJ), is a respiratory virus of mouse and rat first isolated in Sendai, Japan in the early 1950s. The virus is classified as mouse parainfluenza virus type I, belonging to the Paramyxoviridae family. SeV is an enveloped virus, 150-250 nm in diameter, whose genome is a single chain of (-) sense RNA (15,384 bases). The virus infects cells by attaching to the sialic acid receptor present on the surface of many different cells and is thus able to infect a wide range of cell types of various animal species.
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Yes, the additional Klf4 vector allows the system to be fine-tuned by the user. The amount of Klf4 can be increased to enhance reprogramming efficiency, or decreased to minimize the total amount of virus.
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The polycistronic configuration of KOS requires that additional polymerase activity be available to compensate for the combination of three genes on one vector. The Invitrogen CytoTune-iPS 2.0 system uses the extra polymerase from the hKlf4 vector to drive reprogramming in all vectors and enhance reprogramming efficiency. In addition, increased expression of hKlf4 also enhances reprogramming efficiency.
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KOS is an acronym for the genes hKlf4, hOct3/4, hSox2. This is a polycistronic vector, meaning all three of these genes are on one vector.
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The Invitrogen CytoTune-iPS 2.0 Kit (Cat. Nos. A16517, A16518) offers higher reprogramming efficiency, faster clearance of the vectors, and lower cytotoxicity compared to the original Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) that has been discontinued.
The 2.0 kit contains three vectors, one of which is a polycistronic vector (Invitrogen CytoTune 2.0 KOS), designed to deliver increased reprogramming efficiency. This polycistronic vector has a different backbone containing temperature-sensitive mutations in the polymerase-related genes, and this helps to clear the virus faster after reprogramming and causes less cytotoxicity to the cells.
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The Invitrogen CytoTune -iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This kit utilizes four Sendai virus-based vectors, each capable of expressing one of the four Yamanaka factors: Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs.
Please note that the Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued and replaced by the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518).
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Yes we did, and found that only the combination of KOS (3 in 1), cMyc, and Klf4-vector yields highest reprogramming efficiency. For instance, KOS and cMyc alone are not sufficient for reprogramming. Addition of Oct4 or Sox2 results only in a very few reprogrammed colonies. This is mostly due to an imbalance in the stoichiometry of the reprogramming factors, which may impair the reprogramming efficiency significantly.
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The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) is a non-integrating system that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen CytoTune 2.0 reprogramming vectors, including the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs. Only one application of the vectors is required for successful reprogramming.
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The full length Human FGF-basic (FGF-2/bFGF) (aa 1-155) Recombinant Protein is recommended for stem cells whereas the truncated variant, Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein which is missing the first 9 amino acids, is recommended for use with neural and cardiac cells.
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Induced pluripotent stem cells (iPS or iPSCs) are pluripotent stem cells directly generated by introducing combination of genes coding for “reprogramming factors” into adult cells. These reprogramming factors include Oct4, Sox2, c-Myc, KLF4, NANOG, and LIN28. Yu, et al, generated iPS from a human mesenchymal cell line using lentiviral vectors carrying Oct4, Sox2, NANOG, and LIN28 genes (Science 318:1917 (2007)). Using a similar approach, Takahashi et al, generated iPS from human primary fibroblast cells by introducing genes coding for Oct3, Sox2, KLF4, and c-Myc into these cells (Cell 131:861 (2007)). iPS generated by reprogramming are similar to human ES cells in morphology, the capacity for unlimited proliferation, surface-antigen expression, gene expression, the ability to differentiate into cell types representing the three germ layers in vitro, and the ability to form teratomas after injection into SCID mice.
We have seen no effect on reprogramming efficiency as compared to Gibco Essential 8 Medium with Invitrogen CytoTune -iPS 2.0 Sendai Reprogramming Kit. We do recommend daily feeding during colony formation (days 7 - 28).
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